Using aqueous two-phase flotation (ATPF), a new pretreatment method for separating and concentrating liquiritin and glycyrrhizic acid from the aqueous extract of Glycyrrhiza uralensis Fisch was developed, and the target compounds were obtained with high purity by further purification of multi-stage preparative high performance liquid chromatography (pre-HPLC). In the ATPF section, the effects of flotation solvent, solution pH, (NH4)(2)SO4 concentration in aqueous solution, N-2 flow rate and flotation time on the recovery were investigated in detail, and the optimal conditions of ATPF were selected. The emulsification effect in ATPF process was discovered for the first time, and the significant influence on the recovery of target compounds was discussed in detail. Under the optimal conditions (PEG2000 as the flotation solvent, pH 7, (NH4)(2)SO4 concentration of 350 g/L in aqueous phase, N-2 flow rate of 50 ml/min, and flotation time of 40 min), the recoveries of liquiritin and glycyrrhizic acid were more than 93% and 88%, respectively. After ATPF concentration, the flotation products were purified by two-step pre-HPLC. As determined by HPLC, both the purities of the final products were over 99%. The research results indicated that the combination of ATPF and pre-HPLC is a feasible and practical method to obtain the water-soluble active compounds from natural product. (C) 2014 Elsevier B.V. All rights reserved.